Analysis and assay of glycated haemoglobins by capillary electrophoresis, buffer compositions and kits for capillary electrophoresis

ABSTRACT

The invention relates to a method for analysis by capillary electrophoresis of glycated haemoglobins comprising at least one globin chain comprising a glucose residue bound to the amino acid in the N-terminal position, contained in a biological sample, said method comprising using a buffer composition comprising at least one compound which is capable of specifically complexing glucose residues of one or several glycated haemoglobin(s) and of providing said glycated haemoglobin(s) with several negative electric charges at an alkaline pH. By way of example, this compound may be 3,4- or 3,5-dicarboxyphenylboronic acid, preferably 3,5-dicarboxyphenylboronic acid. 
     Said method may in particular be used to separate and assay haemoglobin HbA 1c  present in a biological sample optionally comprising other haemoglobins, in particular other minor fractions. 
     The invention also concerns buffer compositions for use in said analysis, as well as kits for the analysis and for the assay of glycated haemoglobins by capillary electrophoresis.

The present application concerns the field of the analysis and assay ofglycated haemoglobins, in particular haemoglobin A_(1c), by capillaryelectrophoresis.

The invention pertains to a method for analysis by capillaryelectrophoresis of a biological sample comprising one or severalhaemoglobin(s) and in particular one or several glycated haemoglobin(s),as well as appropriate buffer compositions for said analysis, and kitsfor analysis and for assay of glycated haemoglobins by capillaryelectrophoresis.

Haemoglobin (Hb) is a globular molecule generally comprising foursub-units each constituted by a polypeptide chain conjugated to a hemeportion. The polypeptide chains are collectively designated by the name“globin portion” of the haemoglobin molecule.

All human haemoglobins are constituted by four polypeptide chains in twogroups of two identical chains. In an adult human, three types ofhaemoglobin are normally present: haemoglobin A (HbA), which is the vastmajority (it generally represents approximately 97% of the haemoglobinin an adult), constituted by 2 alpha chains and two beta chains(haemoglobin α₂β₂), haemoglobin A2 (HbA2, which generally representsapproximately 1-3% of the haemoglobin in an adult) constituted by twoalpha chains and two delta chains (haemoglobin α₂δ₂), and foetalhaemoglobin (HbF), constituted by two alpha chains and two gamma chains(haemoglobin α₂γ₂), which only subsists in trace amounts (generally lessthan 1% of the haemoglobin in an adult) and remains limited to arestricted cell population (“F cells”).

Haemoglobin contains species known as minor species; they are glycatedhaemoglobins.

Glycated haemoglobin (also known as glycosylated haemoglobin orglycohaemoglobin) corresponds to the set of haemoglobin moleculesmodified by binding of oses, principally glucose, on the amine functions(NH₂) of the globin; the NH₂ group of a haemoglobin condenses with analdehyde group derived from a reducing sugar. This reaction, which isnon-enzymatic, is then stabilized by an Amadori rearrangement. Potentialglycation sites are the N-terminal amino acids of the four globin chainsof haemoglobin (α chains and β, δ, or γ chains depending on the type ofhaemoglobin) and the free amino-epsilon groups (in particular those ofthe lysine residues) in the globin chains of the haemoglobin.

Many forms of glycated haemoglobin exist; they depend on the number ofbound oses, the nature of the globin chains and the oses bound on saidchains and on the position of said oses on the globin chains.

The term “total glycated haemoglobin” is used when considering themolecules of glycated haemoglobin on any NH₂ residue.

The term “haemoglobin A1 (HbA1)” is reserved for molecules ofhaemoglobin A having one molecule of ose bound to the N-terminal aminoacid of one or two beta chains of the protein. The A1 fraction, which isheterogeneous, comprises in particular the minor fractions A_(1a),A_(1b) and especially A_(1c). Haemoglobin A_(1a) (HbA_(1a)) includeshaemoglobin A_(1a1) (HbA_(1a1)), in which the ose bound to theN-terminal amino acid is fructose 1-6 diphosphate, and haemoglobinA_(1a2) (HbA_(1a2)), in which the ose bound to the N-terminal amino acidis glucose-6-phosphate. In the case of haemoglobin A_(1b) (HbA_(1b)),the ose bound to the N-terminal amino acid is pyruvate. Finally, the osebound to the N-terminal amino acid of the beta chains of haemoglobinA_(1c) (HbA_(1c)) is glucose; HbA_(1c) is obtained by condensation ofone molecule of glucose with the NH₂ group of the N-terminal valine ofthe β chain, to form a stable keto-amine, derived from a rearrangement(Amadori rearrangement) of a previously formed unstable aldimine(designated as labile HbA_(1c) or preA_(1c)).

Finally, the term haemoglobin A₀ (HbA₀) generally encompassesnon-glycated A haemoglobins and glycated A haemoglobins not comprisingan ose bound to the amino acid in the N-terminal position of the βchains.

Protein glycation (including glycated haemoglobin and in particularhaemoglobin A_(1c), among many others) is caused by a too highconcentration of sugars in the blood (as is the case with diabetes). Theglycation of proteins alters their functions, causing cell lesions,tissue lesions and vascular ageing, and participates in the developmentof several diseases such as arteriosclerosis, renal insufficiency,diabetic retinopathy and cataracts. Once glycated, haemoglobintransports oxygen less well.

Included among all of the groups and sub-groups of haemoglobin, HbA_(1c)is of particular interest as it serves as a long-lasting marker of thediabetic state of patients. The formation of HbA_(1c) is dependent onglycaemia (concentration of glucose in the blood). It intervenesthroughout the lifespan of the red blood cells, which is normally 120days. The blood concentration of HbA_(1c) thus indicates the averageglycaemia over the 2-3 months preceding assay. Elevation of the bloodconcentration of HbA_(1c) is indicative of prolonged hyperglycaemiaduring that period. The blood concentration of HbA_(1c) is notinfluenced by short-term fluctuations in the levels of blood glucose.Thus, by measuring the HbA_(1c) level between two dates, the evolutionof diabetes can be monitored.

In a diabetic patient, the aim is to obtain a HbA_(1c) level that is asclose as possible to the figures characterizing a good balance ofglycaemia. The reference value in a non-diabetic human with a normalglycaemia is 4% to 6% HbA_(1c) in the blood compared with the totalconcentration of A haemoglobins (glycated haemoglobins and non-glycatedhaemoglobins) (i.e. 20 to 42 mmol/moles; Panteghini et al, 2007).

The detection of glycated haemoglobins, in particular the detection ofan increase in the HbA_(1c) level, is thus of manifest interest for thediagnosis of diabetes (type 1 or type 2) or monitoring diabeticpatients, in particular to evaluate the efficacy of a treatment againstdiabetes (type 1 or type 2).

However, interpreting the results may occasionally be difficult, sincemany factors may falsify the results, in particular the presence ofabnormal haemoglobins.

Indeed, haemoglobins termed “abnormal” or variant, in particular denotedby the letters S, C, D, E, H and J, are encountered in certain patients.Haemoglobin A is partially or completely replaced by one or severalabnormal haemoglobins. They originate in particular from reducedsynthesis of certain globin chains and/or from a modification of thestructure of the α, β, γ or δ chains, by substitution of one amino acidby another. Thus, a modification in the β chain may, for example, giverise to S or C haemoglobins (α₂β₂ haemoglobins in which the glutamicacid in position 6 in the β chain is respectively substituted by avaline or a lysine).

These abnormal haemoglobins are also susceptible to glycation. Thus,haemoglobins S_(1c) (HbS_(1c)) and C_(1c) (HbC_(1c)) exist which, likeHbA_(1c), comprise one molecule of glucose bound to the N-terminalvaline on the β chains (Abraham et al, 1984).

Methods for analysis and assay of glycated haemoglobin and in particularhaemoglobin HbA_(1c) may be classified into 2 broad categories.

The first category, the major category, includes methods such asimmunoassay or affinity chromatography. It is based on the structuralcharacteristics of the sugar molecule bound to the haemoglobin. As anexample, the publication by Wilson et al, 1993 and U.S. Pat. No.6,162,645 describe a method for the assay of glycated haemoglobin foundin a sample of human blood by affinity chromatography. Said method isbased on the use of a solid positively charge phase, coupled via apolyanionic compound (for example polyacrylic acid), to boronic acid,phenylboronic acid, boric acid or a similar boronate compound. When ablood sample to be analyzed is incubated with that solid phase, theglucose residues of the glycated haemoglobin molecules present in thatsample are complexed with the boronate compound. The glycatedhaemoglobin molecules are thus grafted onto the solid phase. Theproportion of glycated haemoglobin immobilized on the solid phasecompared with the proportion of non-immobilized haemoglobin can then bequantified. That assay is carried out either by measuring the extinctionof fluorescence (Wilson et al, 1993), or by using labelled antibodiesdirected against human haemoglobin (U.S. Pat. No. 6,162,645).

The second category of methods for the analysis and assay of glycatedhaemoglobin includes ion exchange chromatography or electrophoresis. Itexploits the physico-chemical characteristics of the molecules and isbased on the charge difference existing between glycated proteins andnon-glycated proteins.

Of the various analytical methods allowing glycated haemoglobin to beassayed that are described in the literature, electrophoretic methodsare divided into several categories: gel analyses include thepolyacrylamide gel isoelectric focusing analysis (Beccaria, 1978; Simon,1982; Stickland, 1982; Bosisio, 1994) and agarose gel analysis (U.S.Pat. No. 5,246,558; U.S. Pat. No. 4,222,836; Menard, 1980). Capillaryelectrophoresis analyses include isoelectric focusing analysis (Molteni,1994; Hempe, 1994), free solution analysis using an anti-A_(1c) antibody(U.S. Pat. No. 5,431,793), free solution analysis using a nakedcapillary (U.S. Pat. No. 5,599,433) and free solution analysis using adynamic coating (EP 0 733 900 A2; U.S. Pat. No. 5,611,903).

Analysis by electrophoresis of a biological sample can separate thevarious proteins present in the sample, in particular the varioushaemoglobins, and enables determination of the quantity and/orproportion of one or several protein(s) of interest in the biologicalsample. In capillary electrophoresis, in a capillary filled with anelectrolyte, the proteins of a biological sample migrate under theeffect of an electric field from the anode towards the cathode as afunction of their mass and their charge. They thus produce anelectrophoretic migration profile comprising a series of peaks (alsotermed fractions), each of them corresponding to one or severalproteins. Abnormal haemoglobins such as HbS, HbC and HbE have adifferent electrophoretic mobility from that of HbA. Further, because ofthe ose residue bound to the N-terminal amino acid of the beta chains,the HbA₁ has a reduced isoelectric point and, as a result, an electriccharge that is slightly different from that of the other HbA₀ typehaemoglobins. Thus, in an electrophoretic field or in an ion exchangeresin, the migration rate of HbA₁ is different from that of HbA₀, whichenables separating HbA₁ from HbA₀, which has a different charge.

The advantage of capillary electrophoresis also resides in the fact thatonly very small quantities of the biological sample to be analyzed arenecessary. Further, separation by this technique may be very rapid,since high voltages can be used without causing the sample to heat uptoo much during separation.

Analysis by isoelectric focussing (Molteni, 1994; Hempe, 1994) iscarried out in coated capillaries. Methylcellulose is used for thecoating. The catholyte used is sodium hydroxide (NaOH) and the anolyteis phosphoric acid (H₃PO₄). By using ampholytes, this analysis enablesseparation of various variants of haemoglobin, including HbA_(1c), thepeak of which is very close to that of HbA (ΔpI<0.10). Although thismethod functions well, it is not simple to carry out routinely andnecessitates great precision, in particular as regards the pH range ofthe ampholytes (the isoelectric point (pI) of HbA_(1c) is very close tothat of HbA₀).

Free solution analysis using an anti-HbA_(1c) antibody (U.S. Pat. No.5,431,793) is carried out in a borate buffer at a basic pH (pH of 8 to10), using samples subjected to a reaction with a anti-HbA_(1c)monoclonal antibody. More precisely, a first electropherogram isproduced using the working sample without prior reaction with ananti-HbA_(1c) antibody. A single peak is obtained, with area x,containing all of the haemoglobins (including HbA_(1c)). A secondelectropherogram is obtained by analyzing the working sample which hasbeen brought into contact with the anti-HbA_(1c) antibody. The non-boundanti-HbA_(1c) antibody, the anti-HbA_(1c) antibody/HbA_(1c) complex andthe haemoglobins not complexed by the antibody are thus separated. Thequantity of HbA_(1c) is then determined by the difference of the areaunder the peaks between the peak for the first electropherogram and thatof the second relative to the haemoglobins not complexed by theanti-HbA_(1c) antibody, with area y, i.e. a quantity x-y. This methodthus proves to be long and complicated in implementation.

The free solution analysis method using a naked capillary (U.S. Pat. No.5,599,433) involves a sugar complexing agent bound to haemoglobin(borate) and a zwitterionic buffer (CAPS) at a basic pH (pH of 9 to 12).The profile obtained (shown in FIG. 1 of the present application) underthe conditions described show a rather small separation of the peaks forHbA₁ (described as being the peak for HbA_(1c) in patent U.S. Pat. No.5,599,433) and HbA₀ (see Example 1). Further, analysis of the purifiedminor fractions shows that the HbA_(1a,b) fractions co-migrate withHbA_(1c), interfering with the final assay result (see Example 2 andFIG. 2 of the present application). In fact, that technique separatesthe HbA₀ and HbA₁ fractions, but does not separate the HbA₁ fractionsthemselves.

The technique for capillary analysis with a dynamic double coating (EP 0733 900 A2; U.S. Pat. No. 5,611,903) is used in the only test based oncapillary electrophoresis which is commercially available (AnalisCEofix™ HbA_(1c) kit). It consists of a first wash of the capillary withan “initiator” containing a polycation (in solution, pH 4.6) resultingin coating the wall of the capillary with said polycation. A second washis then carried out with the analysis buffer containing a polyanion (pH4.6), which has the effect of providing a second coating layer byinteraction with the polycation. The quantity of negative electricalcharges then present on the internal wall of the capillary is evenhigher than with a naked capillary, resulting in a much higherelectroosmotic flux. The resolution obtained between the various formsof haemoglobin analyzed (including glycated Al haemoglobins) issatisfactory, with short analysis times. From a practical viewpoint,this double coat must be re-applied between each analysis using aprecise procedure provided by the kit's manufacturer but which is onlydescribed for a mono-capillary apparatus ((P/ACE, Beckman) not adaptedto routine analyses.

Thus, there is a need for reagents and a simple method of implementationthat can effectively separate glycated haemoglobins, in particularHbA_(1c), from other haemoglobins, variants, interfering forms (labileforms, acetylated forms, carbamylated forms) and from other minorfractions (especially HbA_(1a) and HbA_(1b)) present in a biologicalsample containing other haemoglobins. Ideally, such a method can producea semi-quantitative or quantitative assay of glycated haemoglobins, inparticular of HbA_(1c), directly from the electrophoretic profileobtained.

The present invention proposes an alternative capillary electrophoresismethod, which enables performing a semi-quantitative or quantitativeanalysis of one or several glycated haemoglobin(s) comprising at leastone globin chain comprising a glucose residue bound to the amino acid inthe N-terminal position, contained in a biological sample in particularcomprising other haemoglobins (glycated and/or non-glycated). Saidmethod of the invention exploits both the structural characteristics ofthe glucose molecules bound to said glycated haemoglobins and the chargedifferences existing between the various haemoglobins of the sample.

The inventors have shown that by using a particular buffer composition,it is possible to obtain greatly improved separation of glycatedhaemoglobin(s) comprising a glucose residue bound to the amino acid inthe N-terminal position on at least one globin chain, and in particularon the beta chains, and more particularly a greatly improved separationof HbA_(1c).

One of the advantages of the method of the invention is that becausesaid buffer composition is used, the electrophoresis peak correspondingto one or several types of glycated haemoglobin(s) of interest (one orseveral types of haemoglobin(s) comprising a glucose residue bound tothe amino acid in the N-terminal position on at least one globin chain,for example HbA_(1c)) is displaced, when it is present in a biologicalsample, with respect to the position of the peak which would be obtainedwithout the buffer composition. This displacement does not interferewith the separation of the other proteins, in particular the otherhaemoglobins (glycated and/or non-glycated) of the sample. This inparticular enables separation of HbA_(1c) from other minor HbA₁ (inparticular HbA_(1a) and HbA_(1b)). As a consequence, means that theresults of the analysis can be read reliably and allows the accurateassay of one or several glycated haemoglobin(s) of interest comprisingone or several glucose residue(s) with one glucose bound to the aminoacid in the N-terminal position of at least one globin chain (forexample haemoglobin A_(1c)).

The method of the invention thus constitutes a method of choice with aview to establishing a diagnosis or of monitoring diabetic patients, inparticular to evaluate the effectiveness of an anti-diabetic treatment.

Thus, the present application provides a method for analysis, bycapillary electrophoresis, of glycated haemoglobins (one or severalglycated haemoglobin(s)) comprising one or several glucose residue(s)contained in a biological sample comprising one or severalhaemoglobin(s), said method comprising using a buffer compositioncomprising at least one compound which is capable of specificallycomplexing glucose residues (one or several residue(s)) of glycatedhaemoglobins (one or several glycated haemoglobin(s)) of the biologicalsample and also of providing said glycated haemoglobins with severalnegative electrical charge(s) at an alkaline pH (i.e. a pH of more than7).

The term “several” as used in the context of the present applicationmeans at least two, i.e. two or more than two, for example, two, three,four, five, six, seven, eight, nine, ten or more than ten.

The term “at least one” as used in the context of the presentapplication means one or several.

The term “haemoglobin” as used in the context of the present applicationmeans any form or fraction of haemoglobin (including the minorfractions), whether they be normal or abnormal, as well as the manyvariants of said haemoglobins (at least 1000 variants of haemoglobinhave been described).

The term “glycated haemoglobin” as used in the context of the presentapplication means any haemoglobin modified by binding one or severalsugar(s), in particular one or several glucose, glucose-6-phosphate,fructose 1-6 diphosphate or pyruvate, to one or several globin chain(s)(whatever the globin chain). The sugar(s) may be bound to the amino acidin the N-terminal position on a globin chain and/or to an amino acidwith a free amino acid group (for example lysine).

The term “globin chain” as used in the present application means anyglobin chain known to the skilled person, in particular a chain selectedfrom alpha (α), beta (β), delta (δ) and gamma (γ) chains, whether normalor abnormal, or a variant of one of said chains. According to aparticular embodiment, said “globin chain” is a beta chain, for examplea beta chain of a A₁ haemoglobin, in particular A_(1c), S or C.

When reference is made in the embodiments of the invention to “glycatedhaemoglobin”, it should be understood that said embodiments are ofparticular application to each particular type of glycated haemoglobincited in the present application.

Unless otherwise indicated, each embodiment indicated in thisapplication is applicable independently of and/or in combination withany or several of the other described embodiments.

The capillary electrophoresis analysis method of the invention isapplicable to a biological sample comprising at least one glycatedhaemoglobin comprising one or several glucose residue(s) and necessarilycomprising, on one or several globin chain(s) and in particular on thebeta chains, a glucose residue bound to the amino acid in the N-terminalposition. These haemoglobins are designated as “haemoglobins glycated inthe N-terminal position” in the present application.

Said method enables separation of one or several haemoglobins glycatedin the N-terminal position from other glycated haemoglobins (i.e.glycated haemoglobins wherein the globin chains are devoid of a glucoseresidue bound to the amino acid in the N-terminal position) or fromnon-glycated haemoglobins, which may be present in the analyzedbiological sample.

According to a particular embodiment, the amino acid in the N-terminalposition on the beta chain(s) is valine.

In a preferred embodiment, the analysis method of the invention isintended for the analysis of a biological sample comprising haemoglobinA_(1c). In particular, it is appropriate for separating haemoglobinA_(1c) from other haemoglobins or from the other haemoglobins(differently glycated or non-glycated) which may be present in theanalyzed biological sample, and in particular from other minorfractions, for example HbA_(1a) and from HbA_(1b).

According to a particular embodiment, the analysis method of theinvention can separate haemoglobin S_(1c) and/or haemoglobin C_(1c) fromthe other haemoglobins (differently glycated or non-glycated) which maybe present in the analyzed biological sample.

The term “buffer composition” means a composition, in particular asolution, which retains approximately the same pH despite the additionof small quantities of an acid or a base or despite dilution.

The term “to complex” or “complexing” is used in the present applicationto signify that an association (chemical or simply physical) occursbetween two entities, in particular between two molecules (for example asmall molecule and a macromolecule) via functional groups.

In a preferred embodiment, one of these two entities (the compound whichis capable of complexing glucose residues) combines with (for examplereacts with) a cis-diol group, in particular with two vicinal hydroxylgroups of a glucose residue from the other entity (a glycatedhaemoglobin).

As can be seen in the “examples” section, “specifically complexing theglucose residue(s) of one or several glycated haemoglobin(s) andproviding said glycated haemoglobin(s) with negative charges at analkaline pH” in the present application means that the compound used inthe context of the present invention allows to displace theelectrophoretic migration peak corresponding to a haemoglobin glycatedin the N-terminal position by glucose, beyond the electrophoreticmigration zone corresponding to haemoglobins glycated in the N-terminalposition by another sugar (for example glucose-6-phosphate, fructose 1-6di-phosphate, or pyruvate), and preferably outside the zone of theelectrophoretic migration peaks corresponding to other haemoglobins ofthe sample. Thus, the minor fractions which do not comprise any glucoseresidue bond to the N-terminal amino acid of the beta chains, inparticular the A_(1a) and A_(1b) fractions, do not interfere with theanalysis of the HbA_(1c) according to the method of the invention.

As an example, the capacity of a compound to specifically complexglucose residues of one or several glycated haemoglobin(s) and toprovide negative charges at an alkaline pH in the context of theinvention may be demonstrated using the following test: a referencesample (for example “HbA_(1a), HbA_(1b) mixture”, “A_(1c)” and “A₀” fromExocell, USA), containing various purified fractions of glycatedhaemoglobin and in particular the A₀ and A_(1c) fractions or the A₀,A_(1b) and A_(1a), fractions, is introduced into a capillaryelectrophoresis capillary containing the following buffer composition:200 mM of CHES, 20 mM of putrescine, between 10 and 120 mM (for example30 mM or 50 mM) of test compound, if necessary 2.5 g/l of sodiumchloride, water, and if necessary, a base to adjust the pH to a valuegreater than 9, for example 9.4. Naked capillary free solution capillaryelectrophoresis is then carried out, for example using a Capillaries®(Sebia) apparatus, at a wavelength of 415 nm. Next, the electrophoreticprofile obtained is studied; if the separation between the peakcorresponding to HbA_(1c) and the peak corresponding to HbA₀ (or,depending on the type of sample used, between the peak corresponding toHbA_(1c) and the peaks corresponding to HbA₀, HbA_(1b) and HbA_(1a)) iscomplete, then the test compound is considered to be capable ofspecifically complexing glucose residues and of providing negativecharges at an alkaline pH in the sense of the invention. In contrast, ifthe peak corresponding to HbA_(1c) and that which corresponds toHbA_(1b) (or, depending on the type of sample used, the peakcorresponding to HbA_(1c) and the peaks corresponding to HbA₀, HbA_(1b)or HbA_(1a)), are superimposed or overlap, then the test compound is notconsidered to be appropriate for carrying out the invention.

The concentration of the test compound in the test indicated above isgiven solely by way of indication; if a concentration of 10 to 120 mM oftest compound cannot produce sufficient separation between the peakcorresponding to HbA_(1c) and the peak corresponding to the otherhaemoglobins of the sample in the context of the test indicated above,it is possible to go beyond this concentration range in order to obtainoptimal separation of the peak corresponding to HbA_(1c).

Any chemical compound (i.e. generally an organic molecule), anyantibody, polypeptide, peptide, or any other compound which is capableof specifically complexing glucose residues and of providing negativecharges at an alkaline pH in the sense of the present invention may beused in the context of the present invention. An appropriate antibodymay be generated using any method which is known to the skilled personand may, for example, be a polyclonal or monoclonal antibody, a chimericantibody or a fragment of antibody, for example a Fab fragment.

As can be seen in the “examples” section, boric acid is not a chemicalcompound which is capable of specifically complexing glucose residuesand of providing negative charges at an alkaline pH in the sense of thepresent invention as it does not enable performing a reliable analysisand in particular a reliable assay of glycated haemoglobins and inparticular of HbA_(1c). Examples of appropriate chemical compounds aredefined in more detail below.

Each glucose residue complexed with the compound which is capable ofspecifically complexing glucose residues interacts with a differentmolecule of compound.

According to a particular embodiment, when it is present in sufficientquantity in the buffer composition of the invention, said compound is inparticular capable of specifically complexing the N-terminal glucoseresidue(s) of a glycated haemoglobin, i.e. for each globin chain (inparticular beta) comprising a glucose residue bond to the amino acid inthe N-terminal position, of specifically complexing said glucoseresidue. Said compound is thus capable of specifically complexing eachglucose residue which is bound in the N-terminal position on the betachains of a A_(1c) haemoglobin or of a S_(1c) or C_(1c) haemoglobin.

According to a particular embodiment, when it is present in sufficientquantity in the buffer composition of the invention, said compound iscapable of specifically complexing all the glucose residues of thehaemoglobins glycated in the N-terminal position by a glucose.

According to a particular embodiment, when it is present in sufficientquantity in the buffer composition of the invention, said compound iscapable of specifically complexing all of the glucose residues of ahaemoglobin, regardless of their position on the globin chain(s). Thus,when said compound is present in sufficient quantity in the buffercomposition of the invention, a haemoglobin molecule comprising a numberx of glucose residues can bind a number x of molecules of said compound.

By specifically complexing with one or several glucose residue(s) ofglycated haemoglobin(s) of a biological sample, said compound providessaid glycated haemoglobin or haemoglobins with several negativeelectrical charges at an alkaline pH, i.e. enables surcharging theseglycated haemoglobin(s) with negative electric charges at an alkalinepH.

Said compound provides each glucose residue of the same haemoglobincomplexed thereby with several negative electric charges at an alkalinepH. Because of this provision of negative electric charges at analkaline pH, the electrophoretic mobility of the haemoglobins comprisingat least one glucose residue is modified as a result of their complexingwith said compound. This is manifested on an electrophoretic profile bythe displacement of the electrophoresis peak(s) corresponding to theglycated haemoglobins comprising one or several glucose residue(s) whichare present in the analyzed biological sample.

All of the haemoglobins comprising at least one glucose residue whichare present in the biological sample form a complex with the compoundcomplexing glucose and providing negative electric charges at analkaline pH (of course providing that said compound is present insufficient quantity to complex with each of these haemoglobins).However, the different haemoglobins and in particular the differenthaemoglobins comprising at least one glucose residue all have adifferent isoelectric point (due to the nature of their globin chains,the number and the position of the glucose residue(s) on those globinschains and the number, nature and position of other sugars that may bebound to said haemoglobins). Because of the negative electric charges atan alkaline pH provided by the complexing of each glucose residue withsaid compound, the difference in charge between these varioushaemoglobins is even more marked when they are in the form which iscomplexed with said compound.

The glycated haemoglobins which include a glucose residue bound to theamino acid in the N-terminal position on at least one of their globinchains (in particular on the beta chains) thus have at least one glucosemore than the other haemoglobins (differently glycated or non-glycated).In particular, haemoglobin A_(1c) has at least one more glucose on thebeta chains than the other minor fractions of haemoglobin A₁. As aconsequence, when they are complexed according to the invention, thesehaemoglobins glycated in the N-terminal position are more heavily loadedwith negative electric charges at an alkaline pH by said compound thanthe other haemoglobins (the glycated haemoglobins not comprising aglucose residue bound to the N-terminal amino acid of the globin chainsor the non-glycated haemoglobins).

Thus, by increasing the difference in electrophoretic mobility existingbetween the various haemoglobins comprising glucose and/or between oneor several haemoglobin(s) comprising glucose and the other haemoglobinspresent in the analyzed biological sample, better separation is achievedbetween one or several haemoglobin(s) glycated in the N-terminalposition and the other haemoglobin(s) which may be present in saidsample, in particular between haemoglobin A_(1c) and the other minorfractions of haemoglobin A₁ haemoglobin which may be present in saidsample.

Thus, the method of the invention can effectively separate haemoglobinsglycated in the N-terminal position, in particular HbA_(1c), from otherhaemoglobins, from certain variants and from other minor fractionspresent in a biological sample containing other haemoglobins.Furthermore, conventional interfering molecules such as labile,acetylated or carbamylated forms do not interfere with the assay ofHbA_(1c) using the method of the invention. In particular, the method ofthe invention enables to avoid interferences which may result, in theabsence of negative electric charges at an alkaline pH provided bycomplexing, from the co-migration of haemoglobin A_(1c) and by otherminor fractions of haemoglobin A₁ (including HbA_(1a) and HbA_(1b))present in the biological sample, during the electrophoretic separationstep.

The separated haemoglobin(s) glycated in the N-terminal position maythen be assayed in the presence of other or of the other haemoglobinswhich may be present in the analyzed biological sample. Thus, as anexample, HbA_(1c) may be assayed in the presence of other minorfractions of haemoglobin A₁ and in particular in the presence ofHbA_(1a) and/or HbA_(1b) without these other minor fractions present inthe analyzed biological sample interfering with that assay.

The term “to assay” or “assay” in the present application means that thequantity of haemoglobin or haemoglobins of interest, possibly in thepresence of one or several other haemoglobin(s) of the analyzedbiological sample, is determined and/or the proportion of saidhaemoglobin(s) of interest is determined with respect to the totalquantity of haemoglobin or with respect to the quantity of certainhaemoglobins present in said sample.

The assay carried out in the context of the invention may besemi-quantitative; thus, the percentage of a given haemoglobin ismeasured with respect to the quantity of another haemoglobin or theother haemoglobin(s) present in said sample. Thus, in the case ofHbA_(1c), in general the percentage of HbA_(1c) is measured with respectto the quantity of one or several other A haemoglobin(s); in general,the area of the electrophoretic peak corresponding to HbA_(1c) isdivided by the area of the peak corresponding to HbA₀ or by the sum(area of peak corresponding to HbA_(1c)+area of peak corresponding toHbA₀), by the sum (area of all of the HbA peaks) or by the sum (area ofpeak corresponding to HbA_(1c)+area of peak corresponding to HbA₀+areaof peak corresponding to HbA₂), or even by the total surface area of theelectrophoretic profile.

The assay may also be quantitative; the results are then expressed inmillimoles (mmoles) of a given haemoglobin per mole of anotherhaemoglobin or other haemoglobin(s) present in said sample, for examplein mmoles of HbA_(1c) per mole of HbA.

According to a particular embodiment of the invention, the compoundspecifically complexing glucose residue(s) of glycated haemoglobin(s)and providing said glycated haemoglobins with negative charges at analkaline pH comprises several functional groups, in particular two ormore than two functional groups.

At least one of said functional groups specifically complexes a glucoseresidue (in particular a glucose residue bond to the amino acid in theN-terminal position on a globin chain) by interacting, for example, witha cis-diol group, in particular with two vicinal hydroxyl groups, of aglucose residue. As an example, this(these) group(s) may be a boronategroup, in particular a boronate group as defined in the presentapplication.

Said glucose complexing group(s) may provide glycated haemoglobin withone negative electric charge at an alkaline pH per complexed glucoseresidue, i.e. one or several negative charge(s) at an alkaline pH percomplexed haemoglobin molecule (one negative charge if the haemoglobinmolecule comprises only one glucose residue and n negative charges ifthe haemoglobin molecule comprises n glucose residues).

The other, one of the others, or the other functional group(s) of saidcompound (i.e. the functional group(s) which do not complex withglucose) provide(s) glycated haemoglobin with one or several negativeelectric charge(s) at an alkaline pH for each complexed residue ofglucose of a molecule of said haemoglobin.

Thus, in this particular embodiment of the invention, each molecule ofcompound specifically complexing glucose residues according to theinvention comprises at least one functional group which is capable ofcomplexing a glucose residue of a glycated haemoglobin (and inparticular a N-terminal glucose residue of a haemoglobin glycated in theN-terminal position) and at least one functional group which does notcomplex glucose but enables surcharging said glycated haemoglobin withnegative charges at an alkaline pH since it provides said glycatedhaemoglobin with one or several supplemental negative charge(s) at analkaline pH. The functional group which is capable of complexing glucosemay also provide a negative electric charge at an alkaline pH.

According to a particular embodiment of the invention, the compoundspecifically complexing glucose residues and providing negative chargesat an alkaline pH (more precisely each molecule of said compound)provides a glycated haemoglobin molecule, and in particular to ahaemoglobin glycated in the N-terminal position, with several negativeelectric charges at an alkaline pH, for each complexed residue ofglucose of said glycated haemoglobin molecule. Preferably, for eachcomplexed glucose residue, it provides one glycated haemoglobin moleculewith at least two (in particular two, three, four, five or six) negativeelectric charges at an alkaline pH.

According to a particular embodiment of the invention, the functionalgroup or at least one of the functional groups specifically complexingglucose residues is negatively charged at an alkaline pH.

According to a particular embodiment of the invention, the compoundwhich is capable of specifically complexing glucose residues of glycatedhaemoglobins and of providing said glycated haemoglobins with negativecharges at an alkaline pH comprises one or several groups which areionisable (in particular anionisables) at an alkaline pH. Said compoundmay be of various types. For example, the compound may comprise one orseveral carboxylate(s), carboxyl(s), sulphonate(s) and/or sulphonyl(s).Said compound may in particular be a polycarboxylic acid, in particulara dicarboxylic or tricarboxylic acid. Said compound may also be apolysulphonic acid, in particular a disulphonic or trisulphonic acid.

According to a particular embodiment of the invention, the group, one ofthe groups or the groups providing a glycated haemoglobin molecule withone or several negative electric charge(s) at an alkaline pH for eachcomplexed glucose residue of said glycated haemoglobin moleculecomprises or consists of one or several group(s) which are ionisable atan alkaline pH as defined in the present application, and in particularone or several (in particular two or three) carboxylate, carboxyl,sulphonate and/or sulphonyl group(s).

According to a particular embodiment of the invention, the group, one ofthe groups or the groups specifically complexing glucose residuescomprise or consist of one or several boronate group(s).

In the present application, the term “boronate group”, means a groupwith formula:

in which D₁ and D₂ are selected, independently of each other, from ahydroxyl group and a group susceptible of being hydrolyzed to produce ahydroxyl group in aqueous solution, in particular at an alkaline pH.

In a particular embodiment of the invention, said boronate group is thegroup —B(OH)₂, (also known as a boronyl group) or an ionized form, inparticular —B(OH)⁻ ₃.

In a particular embodiment of the invention, the compound which iscapable of specifically complexing glucose residues of glycatedhaemoglobins and providing said glycated haemoglobins with negativecharges at an alkaline pH is:

-   -   (i) a boronate compound, with general formula RB(OH)₂ (compound        also known as boronic acid), or with general formula RB(OH)⁻ ₃,        wherein the group R comprises at least one aryl and/or an alkyl        (linear, branched or cyclic) and/or an aralkyl and/or a        combination thereof, and said group R provides glycated        haemoglobins with one or several negative electric charge(s) at        an alkaline pH for each glucose residue complexed with the        boronate group; or    -   (ii) a salt of said boronate compound.

Thus, at an alkaline pH, in addition to the negative electric charge atan alkaline pH provided by the boronyl or boronate of the boronatecompound or its salt, the group R provides glycated haemoglobins withone or several supplemental negative electric charge(s) for each glucoseresidue complexed with the boronyl or boronate group.

In the context of the present application, the term “compound withgeneral formula RB(OH)₂″ also means any ionic form in equilibriumtherewith, in particular RB(OH)⁻ ₃, or any form that is capable of beingin equilibrium therewith depending on the conditions of the medium.

In the context of the present application, the term “salt” denotes anysalt and in particular a sodium, lithium or potassium salt.

The group R of the boronate compound of the invention may also includeother functions and/or heteroatoms, in addition to the aryl, alkyl,aralkyl function(s) or one of their combinations.

According to a particular embodiment, for each glucose residue complexedwith the boronyl or boronate group of the boronate compound of theinvention or its salt, the group R provides two or more (preferably two)negative electric charges at an alkaline pH.

According to a particular embodiment, the group R consists of one orseveral aryl(s), alkyl(s) (linear, branched or cyclic) and/or aralkyl(s)and/or a combination thereof.

According to a particular embodiment, the aryl(s), alkyl(s), aralkyl(s)or other functional groups and/or heteroatoms or their combinationspresent in the group R of the boronate compound of the invention may besubstituted.

The term “substituted” as used in the present application may signifymono-substituted or, in contrast, polysubstituted, in particular di-,tri-, tetra-, penta- or hexa-substituted.

The substituents may in particular be groups which are ionizable (inparticular anionisables) at an alkaline pH as defined in the presentapplication. The group R may include one or several heteroatoms, orother functional groups.

According to a particular embodiment of the invention, the group R ofthe boronate compound provides a glycated haemoglobin molecule with oneor several negative electric charges at an alkaline pH for eachcomplexed glucose residue. Preferably, for each complexed glucoseresidue, it provides a glycated haemoglobin molecule with one, two,three or four negative electric charges at an alkaline pH.

A boronate compound as defined above in particular comprises boronicacids, and in particular phenylboronic acids.

According to a particular embodiment of the invention, the compoundwhich is capable of specifically complexing glucose residues of glycatedhaemoglobins and of providing said glycated haemoglobins with negativecharges at an alkaline pH is a polysubstituted phenylboronate, inparticular a disubstituted phenylboronate, for example disubstitutedwith carboxyl and/or sulphonyl groups.

According to a particular embodiment of the invention, the group R ofthe boronate compound or its salt is a diacid, in particular adicarboxylic acid. According to a particular embodiment of theinvention, the boronate compound is a dicarboxyphenylboronic acid,preferably selected from 3,5-dicarboxyphenylboronic acid and3,4-dicarboxyphenylboronic acid.

Thus, by way of example, the boronate compound employed may be3,5-dicarboxyphenylboronic acid (3,5-dCPBA). This compound iscommercially available, in particular from the supplier Combi-blocksInc. (San Diego, USA), under the trade name 3,5-dicarboxyphenylboronicacid and from Apollo Scientific Ltd (Cheshire, United Kingdom), underthe trade name 3,5-dicarboxybenzeneboronic acid.

In the buffer composition of the invention, the concentration of thecompound which is capable of specifically complexing glucose residues ofglycated haemoglobins and of providing said glycated haemoglobins withnegative charges at an alkaline pH is generally in stoichiometric excesswith respect to the total quantity of proteins, in particular withrespect to the total quantity of all of the haemoglobins present in thebiological sample or with respect to the total quantity of all of thehaemoglobins comprising glucose present in the biological sample. Thus,the quantity of this compound in the buffer composition is greater thanthe quantity necessary for all of the glucose residues of thehaemoglobins present in the sample to be complexed by said compound whenthe sample or an aliquot of that sample is diluted in the buffercomposition. Thus, for each molecule of glycated haemoglobin comprisingglucose present in the analyzed biological sample, there are at least asmany molecules of that compound in the buffer composition as there areglucose residues present in this glycated haemoglobin molecule. Thisenables achieving a complete separation of the glycated haemoglobin orglycated haemoglobins of interest from the other haemoglobins alsopresent in the analyzed biological sample.

According to a particular embodiment of the invention, in the buffercomposition of the invention, the concentration of compound which iscapable of specifically complexing glucose residues of glycatedhaemoglobins and of providing said glycated haemoglobins with negativecharges at an alkaline pH is in the range 0.10 to 100 mM, preferably inthe range 10 to 60 mM, and more preferably in the range 20 to 50 mM, forexample 30 mM or 50 mM.

The expression “in the range x to y” as used in the present applicationmeans that the limits x and y are included in the indicated range x-y.

According to a particular embodiment, the buffer composition of theinvention further comprises:

-   -   a buffer compound with a pKa in the range 8.0 to 11.0; and/or    -   a flow retardant; and/or    -   a base; and/or    -   a salt; and/or    -   an appropriate dilution solution, for example water.

Thus, the buffer composition of the invention comprises or consists of(i) a compound which is capable of specifically complexing glucoseresidues of glycated haemoglobins and of providing said glycatedhaemoglobins with negative charges at an alkaline pH, and (ii) one orseveral and in particular all of the compounds selected from: a buffercompound having a pKa in the range 8.0 to 11.0, a flow retardant, abase, a salt, an appropriate diluting solution (for example water) andmixtures thereof.

The buffer compound is preferably a zwitterionic compound. It may inparticular be selected from AMP, AMPD, AMPSO, bicine, CABS, CAPS, CAPSO,CHES, HEPBS, methylamine, TABS, TAPS, taurine, tricine and Tris; inparticular, CAPS, CAPSO or CHES are selected, preferably CHES. Thesecompounds have a high buffering power at the target pH (pH 8-11) and areparticularly suitable for obtaining good focussing of the haemoglobins.

The concentration of buffer compound in the buffer composition of theinvention is generally in the range 20 to 500 mM, preferably in therange 50 to 400 mM, and more preferably in the range 100 to 350 mM or inthe range 150 to 300 mM, for example approximately 200 mM orapproximately 300 mM.

The flow retardant is intended to strengthen the effect of thedifferences in electric charge in order to obtain a good resolution ofseparation between the various haemoglobins. This type of compound actsby reducing the electro-osmotic flux, which retards migration of thevarious fractions and can increase their separation. The flow retardantused may be an aliphatic diamine, in particular an aliphatic diamineselected from 1,3-diaminopropane, 1,4-diaminobutane (putrescine),1,5-diaminopentane (cadaverine), 1-6-diaminohexane, spermine and DETA,or a derivative of said aliphatic diamine or a mixture thereof.

The term “derivative” as used herein means an aliphatic polyamine, acyclic polyamine, a salt (for example a sodium salt) or one of theirmixtures.

According to a particular embodiment of the invention, the flowretardant is selected from putrescine, its derivatives and mixturesthereof.

According to a particular embodiment of the invention, the flowretardant is putrescine. In particular, putrescine is in the pure form,a salt being optionally added.

According to another particular embodiment of the invention, the flowretardant is putrescine hydrochloride (or putrescine-2HCl).

In the buffer composition of the invention, the concentration of flowretardant is advantageously in the range 0.10 to 40 mM, preferably inthe range 10 to 30 mM and more preferably in the range 15 to 25 mM, forexample 20 mM.

The base optionally added to the buffer composition allows the pH ofsaid composition to be adjusted. In particular, a base belonging to thehydroxide family may be used, in particular a base selected from lithiumhydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide,caesium hydroxide, francium hydroxide, a mono-, di-, tri- or tetra-alkylammonium hydroxide, and mixtures thereof.

According to a particular embodiment of the invention, the baseoptionally added to the buffer composition is sodium hydroxide.

Advantageously, sufficient base is added to the buffer composition sothat its pH is 9.0 or more, preferably in the range 9.0 to 11.0 and morepreferably in the range 9.0 to 10.0, for example in the range 9.3 to9.5, and still more preferably a pH of 9.4 or 9.5. An alkaline pH ofmore than 9 can produce negatively charged haemoglobin fractions (theisoelectric point of haemoglobins being in the range 6.05 to 7.63).

According to a particular embodiment of the invention, the salt which isoptionally present in the buffer composition of the invention is asodium salt, preferably sodium chloride. The buffer composition maycomprise for example 2.5 g/l of sodium chloride.

According to a particular embodiment of the invention, when the buffercomposition comprises putrescine, in particular pure putrescine, itfurther comprises a salt (in particular a sodium salt), preferablysodium chloride, for example 2.5 g/l of sodium chloride.

According to another particular embodiment of the invention, the buffercomposition comprises putrescine hydrochloride, does not comprise sodiumchloride and preferably does not comprise any salt.

According to a particular embodiment of the invention, the buffercomposition comprises or consists of:

-   -   200 mM of CAPSO;    -   10 mM of putrescine (for example either putrescine-2HCl or pure        putrescine and sodium chloride);    -   50 mM of 3,5-dicarboxyphenylboronic acid;    -   water; and    -   if necessary, a base, for example sodium hydroxide, to adjust        the pH, for example to a value greater than 9 or 10, and        preferably to a value of 10.2;        or, more preferably, of:    -   200 mM of CAPSO;    -   15 mM of putrescine (for example either putrescine-2HCl or pure        putrescine and sodium chloride);    -   100 mM of 3,5-dicarboxyphenylboronic acid;    -   water; and    -   if necessary, a base, for example sodium hydroxide, to adjust        the pH, for example to a value greater than 9 or 10, and        preferably to a value of 10.2;        or, still more preferably, of:    -   200 mM of CHES;    -   20 mM of putrescine (for example either putrescine-2HCl or pure        putrescine and sodium chloride);    -   30 mM of 3,5-dicarboxyphenylboronic acid;    -   water; and    -   if necessary, a base, for example sodium hydroxide, to adjust        the pH, for example to a value greater than 9, and preferably to        a value of 9.4.

According to another particular embodiment of the invention, the buffercomposition comprises or consists of the components indicated above inthe concentrations indicated above, and one or several salt(s), inparticular a sodium salt, especially sodium chloride, for example 2.5g/l of sodium chloride.

As illustrated in the example section, such buffer compositions enableachieving a perfect separation of haemoglobin HbA_(1c) from the otherhaemoglobins present in the biological sample analyzed, as well asmutual separation of the various minor fractions (including HbA_(1a),HbA_(1b) and HbA_(1c)) in a manner unequalled in the field of freesolution capillary electrophoresis.

The term “biological sample” as used in the present application meansany biological fluid comprising red blood cells. Said biological fluidmay derive from a healthy or sick human patient (for example a diabeticpatient) or be of animal origin, in particular deriving from a non-humanmammal (healthy or sick). Said biological liquid (human or non-humanmammal) may, for example, be blood, in particular normal or non-normalblood, washed, decanted, centrifuged, haemolysed or whole blood. Thebiological samples may also be of synthetic or purified origin.

The invention is of particular application in the analysis of bloodsamples, in particular for the analysis of a blood sample deriving froma diabetic patient or from a diabetic non-human mammal.

According to a particular embodiment, said biological sample comprisesor consists of several haemoglobins, in particular several glycatedhaemoglobins comprising a glucose residue bound to the amino acid in theN-terminal position on at least one globin chain (for example on thebeta chains), and more particularly haemoglobin A_(1c).

The biological sample used may be diluted prior to analysis by capillaryelectrophoresis with a suitable diluting solution, for example ahaemolyzing solution and/or an analyzing buffer solution, in particularthe buffer composition of the invention. Preferably, the analysis iscarried out using a sample of haemolyzed blood.

According to another particular embodiment, the biological sample isinitially diluted in a haemolyzing solution, then in the buffercomposition of the invention.

The term “haemolyzing solution” as used in the present applicationdesignates a solution capable of causing haemolysis of red blood cells,i.e. the destruction of red blood cells, and thus of liberatinghaemoglobin. Depending on its composition, it can carry out total lysisof the red blood cells, optionally by employing a small additionalmechanical action (vortex, stirring, etc). The haemolyzing solution mayinclude the usual additives for cell lysis, such as Triton X100, whichis usually used in a concentration of 1 g/L. The haemolyzing solutionmay also optionally include a compound which is capable of specificallycomplexing glucose residues of glycated haemoglobins and of providingsaid glycated haemoglobins with negative charges at an alkaline pH asdefined in the present application.

By way of example, the haemolyzing solution may be selected from thegroup constituted by the haemolyzing solution Capillaries® Hemoglobin(e)from Sebia, the haemolyzing solution MINICAP® Hemoglobin(e) from Sebia,the haemolyzing solution Hydragel® HbA_(1c) from Sebia, pure water,water supplemented with surfactant, in particular water supplementedwith Triton X100 (for example 1g/L of Triton X100), and mixturesthereof.

According to a particular embodiment, the method for analysis bycapillary electrophoresis of the invention comprises the followingsteps:

-   -   a) introducing the buffer composition of the invention and the        biological sample into an electrophoresis capillary; and    -   b) separating the constituents of said biological sample by        capillary electrophoresis.

These two steps are generally preceded by a step for dilution of thebiological sample, for example in a haemolyzing solution. This dilutionstep may in particular be carried out in the sample support, for examplein a Capillaries® (Sebia) dilution segment or in the MINICAP® (Sebia)sample cup.

In step a), the buffer composition of the invention and the biologicalsample may be introduced separately (simultaneously or not) into thesame electrophoresis capillary, the mixture then being produced in thecapillary. As an example, it is possible to introduce the buffercomposition of the invention first, then the sample into theelectrophoresis capillary.

Alternatively, the biological sample may be introduced into anelectrophoresis capillary in step a) in the form of a mixture with thebuffer composition of the invention.

Depending on the type of capillary electrophoresis apparatus used and asa function of the number of analyses to be carried out, a singlecapillary or several capillaries in parallel are used. When a singlecapillary is used, this capillary is generally used several times insuccession, in order to carry out several analyses. Of course, whenseveral capillaries are used, if necessary several electrophoreticmigrations can be carried out in succession, during which severalcapillaries are used in parallel.

The step for separating the constituents of the biological sample bycapillary electrophoresis in particular consists of applying to thecapillary(ies) an electric field with a sufficient voltage to allow theseparation of one or several glycated haemoglobin(s) of interest fromother proteins and in particular from other haemoglobins which may bepresent in the biological sample.

The conditions for carrying out liquid capillary electrophoresis are theconditions generally used by the skilled person for the steps ofintroducing the sample and the buffer composition into thecapillary(ies) and separating the constituents of the sample byelectrophoretic migration. The electric field applied may, for example,be approximately 400 V/cm. They usually comprise washing the capillarieswith a washing solution, washing with the buffer composition used forthe analysis, optionally diluting the sample one or more times,introducing the sample into the capillary(ies), migration and detection.These steps may be carried out by automated machines.

The conditions for carrying out capillary electrophoresis are, forexample, conditions suitable for using the Capillaries® automatedmachine from Sebia or the MINICAP® from Sebia.

The step for separating the constituents of the biological sample isgenerally followed by a step for detection of one or several protein(s),in particular one or several haemoglobins present in the biologicalsample, and more particularly one or several glycated haemoglobins ofinterest present in the biological sample, for example haemoglobinA_(1c).

This detection step may in particular be carried out by measuring theabsorbance, for example at a wavelength of approximately 415 nm, whichis specific to haemoglobin; the haemoglobins may be analyzed at awavelength of approximately 200 nm, but in order to avoid interferencewith plasma proteins in particular, they are preferably analyzed at awavelength of 415 nm.

In the context of the invention, the results of the electrophoreticseparation may be presented as illustrated in the examples, in the formof electrophoretic profiles generated from a detection signalproportional to the quantity of haemoglobin detected. Thus, the methodof the invention generally further comprises a step for generating anelectropherogram from the detection signal.

As indicated above, when it is present in the analyzed biologicalsample, a haemoglobin of interest may be quantified. To this end, thesurface area of the peak corresponding to said haemoglobin isdetermined.

Thus, said method generally also comprises a step for determining, inparticular from an electropherogram, the quantity of one or severalhaemoglobin(s) present in the biological sample and/or the proportion ofone or several haemoglobin(s) present in the biological sample withrespect to the total quantity of proteins, to the total quantity ofhaemoglobin and/or to the quantity of certain haemoglobins (for examplewith respect to the quantity of HbA or HbA₀) present in the biologicalsample. In particular, the proportion of glycated haemoglobin A_(1c),S_(1c) and/or C_(1c) present in the biological sample with respect tothe total quantity of all of the haemoglobins or with respect to thequantity of certain haemoglobin(s) present in the biological sample, forexample with respect to the total quantity of A, S and/or C haemoglobinrespectively, can be determined.

In one particular embodiment of the invention, this assay may beobtained directly from the electrophoretic profile.

According to a particular embodiment of the invention, the analysismethod also comprises a step for quantification of one or severalhaemoglobin(s) present in the analyzed biological sample with respect toone or several standardized calibrator(s) (for example one or severalreference biological samples); this enables standardization of theresults.

Thus, in order to obtain a high level of precision during assay, eachcapillary is generally calibrated every time the electrophoresisapparatus is started up, using reference biological samples (for examplestandard samples containing known concentrations of different naturaland/or synthetic fractions of glycated haemoglobin, purified ifnecessary). As an example, in order to assay HbA_(1c), at least twocalibrators are generally used, for example a first calibratorcomprising a known high concentration of HbA_(1c) and a secondcalibrator comprising a low known concentration of HbA_(1c). Then, witheach capillary of the electrophoresis apparatus, the quantity ofHbA_(1c) present in each calibrator is measured. This enables toestablish a regression curve (over at least two points) for eachcapillary, and thus the measurements carried out using the variouscapillaries can be normalized. This also enables standardization of theresults obtained with respect to a reference method, by using values forHbA_(1c) determined by this method for the calibrators.

According to a particular embodiment of the invention, the capillaryelectrophoresis carried out is a free solution capillaryelectrophoresis, in particular is a naked capillary(ies) free solutioncapillary electrophoresis.

From the point of view of the materials used for the capillaries, thosethat are normal for capillary electrophoresis are employed. Thespecialist will be able to adapt the nature of the capillary and itssize to the requirements of the analysis.

As an example, in a particular embodiment, the electrophoresiscapillary(ies) is (are) of fused silica.

The present invention also pertains to the use of the capillaryelectrophoresis analysis method according to the invention forseparating one or several glycated haemoglobin(s) comprising glucose,more precisely one or several glycated haemoglobin(s) each comprising,on one or several globin chain(s) (for example on the beta chains), oneor several glucose residue(s) bound to the amino acid in the N-terminalposition, from other proteins, in particular from at least one otherhaemoglobin, and preferably from the other haemoglobins present in abiological sample and, if appropriate, to assay said glycatedhaemoglobin(s).

The present invention also pertains to a buffer composition suitable forcapillary electrophoretic analysis of a biological sample comprisinghaemoglobins and in particular one or several glycated haemoglobin(s),more particularly one or several glycated haemoglobin(s) comprising oneor several glucoses on one or several globin chains (for example on thebeta chains). Said buffer composition comprises at least one compoundthat is capable of specifically complexing glucose residues of glycatedhaemoglobins and also of providing said haemoglobin(s) with severalnegative electric charges at an alkaline pH.

Said buffer composition is in particular that used to carry out themethod for capillary electrophoresis of a biological sample presented inthe present application.

In particular, according to a particular embodiment of the invention,the buffer composition comprises a polycarboxylic boronic acid, inparticular a dicarboxylic acid or a tricarboxylic acid, for example3,5-dicarboxyphenylboronic acid, as a compound which can specificallycomplex glucose residues and provide negative electric charges at analkaline pH.

According to another particular embodiment of the invention, the buffercomposition comprises, in addition to the compound which is capable ofspecifically complexing glucose residues and of providing negativeelectric charges at an alkaline pH,

-   -   a flow retardant; and/or    -   a buffer compound with a pKa in the range 8.0 to 11.0; and/or    -   a base; and/or    -   a salt (in particular a sodium salt, for example sodium        chloride); and/or    -   an appropriate diluting solution, for example water.

These various constituents may be as defined in the present application.

According to a particular embodiment of the invention, the buffercomposition has a pH of 9.0 or more, preferably a pH in the range 9.0 to11.0 and more preferably a pH in the range 9.0 to 10.0, for example a pHin the range 9.3 to 9.5, and still more preferably a pH of 9.4. Such apH may in particular be obtained by providing a sufficient quantity of abase as defined above.

The buffer compositions of the invention are prepared in the usualmanner for analytical buffer compositions, namely by providing theconstituents in the liquid form or in the solid form to be diluted, toan acceptable support. Usually, the support is water, distilled ordemineralized.

The present invention also concerns a kit comprising a buffercomposition of the invention and, if appropriate, instructions for use,to carry out electrophoretic analysis. In other words, the kit of theinvention may comprise or consist of a buffer composition of theinvention and a packaging material and, if appropriate, instructions foruse.

Thus, the kit of the invention in particular comprises a compound whichis capable of specifically complexing glucose residues of glycatedhaemoglobin and of providing negative charges at an alkaline pH asdefined in the present application. When, in addition to said compound,said kit comprises other compounds, in particular one or severalcompound(s) selected from: a buffer compound with a pKa in the range 8.0to 11.0, a flow retardant, a base, a salt (in particular a sodium salt,for example sodium chloride), an appropriate diluting solution (forexample water) and mixtures thereof, the various compounds of said kitmay be packaged for extemporaneous mixing, or, in contrast, may bepackaged together, in particular in the same composition, in the form ofa mixture. Alternatively, certain compounds of said kit may be packagedseparately while others may be packaged together, in particular in theform of a mixture.

According to a particular embodiment of the invention, the buffercomposition of the invention is provided in one or several parts, to bereconstituted by the consumer before the analysis. Hence, problems withstability which might arise for example when all of the components orsome components of said composition are packaged in the form of amixture can be overcome.

The kit of the invention may also comprise one or several solution(s)for washing the capillaries and/or one or several dilution segment(s)and/or one or several solution(s) suitable for diluting the biologicalsample to be analyzed (for example a haemolyzing solution, in particulara haemolyzing solution as defined in the present application) and/or oneor several reference biological sample(s) (for example natural and/orsynthetic glycated fractions, purified if necessary) which enable(s)calibrating each capillary.

The present invention also pertains to the use of a buffer compositionas defined in the present application and/or to a kit of the inventionfor the analysis, by capillary electrophoresis, of (one or several)glycated haemoglobins comprising one or several glucose residue(s), inparticular the A_(1c), S_(1c) and C_(1c) haemoglobins contained in abiological sample comprising one or several haemoglobin(s).

The present invention also pertains to the use of a buffer compositionas defined in the present application and/or a kit of the inventionand/or a compound which is capable of specifically complexing glucoseresidues (one or several) of glycated haemoglobins (one or severalglycated haemoglobin(s)) and of providing this or these glycatedhaemoglobin(s) with several negative electric charges at an alkaline pHto separate, by capillary electrophoresis, one or several glycatedhaemoglobin(s) comprising a glucose residue bound to the amino acid inthe N-terminal position on at least one globin chain (for example on thebeta chains), and more particularly to separate, by capillaryelectrophoresis, haemoglobin A_(1c) from other proteins, in particularfrom at least one other haemoglobin and preferably from the otherhaemoglobins present in a biological sample and, if appropriate, toassay said haemoglobin(s) separated thereby.

In particular, the buffer composition of the invention, the kit of theinvention and/or a compound which is capable of specifically complexingglucose residues of glycated haemoglobins and of providing negativeelectric charges at an alkaline pH as defined in the present applicationare appropriate for use, for example in the context of a method of theinvention for diagnosing diabetes in a human or non-human mammal and/orfor monitoring the glycaemic balance of a human or non-human mammal (inparticular a diabetic subject), in particular to evaluate the efficacyof a treatment against diabetes and/or to adapt a diabetes treatment ina diabetic subject. The biological sample(s) analyzed then originatefrom said human or non-human mammal.

The term “diabetes” as used in the present application designates type 1and/or type 2 diabetes.

The present invention also concerns the use of a buffer composition ofthe invention, of a kit of the invention and/or of a compound which iscapable of specifically complexing glucose residues of glycatedhaemoglobins and of providing negative charges at an alkaline pH asdefined in the present application, for the manufacture of a diagnostickit. Said diagnostic kit may in particular be used for the diagnosis ofdiabetes in a human or a non-human mammal and/or to monitor theglycaemic balance of a human or non-human mammal, in particular adiabetic subject. Said kit may thus allow the efficacy of a treatmentagainst diabetes in a human or a non-human mammal suffering fromdiabetes to be evaluated and/or allow a treatment against diabetes in adiabetic subject to be adapted.

The HbA_(1c) level is generally in the range 4% to 6% (i.e. 20 to 42mmoles of HbA_(1c) per mole of haemoglobin in the blood) in a humannon-diabetic, and more than 7% in a diabetic patient (in the absence oftreatment).

In the case of HbA_(1c) levels in the range 6% to 7% (i.e. aconcentration of 42 to 53 mmoles of HbA_(1c) per mole of haemoglobin inthe blood), it is recommended that an anti-diabetic treatment becommenced.

Beyond a threshold of 8%, which is equivalent to a concentration of 64mmoles of HbA_(1c) per mole of haemoglobin in the blood (Panteghini andJohn, 2007), the patient runs an increased risk of developing one of thecomplications of diabetes (microangiopathy, macroangiopathy, etc). It isthen recommended that the patient's anti-diabetic treatment be modified.

Other characteristics and advantages of the invention will becomeapparent from the following examples and figures which illustrate theinvention.

DESCRIPTION OF THE FIGURES

FIG. 1: electropherogram obtained on CE Agilent from a normal humanblood sample using the analysis buffer described in patent U.S. Pat. No.5,599,433, which contains 100 mM of CAPS and 300 mM of boric acid (pH:11.00; temperature: 24° C.; voltage: 6.1 kV, i.e. 190 V/cm; injection:50 mbars 20 s).

FIG. 2: standard electrophoretic profiles for A_(1a,b) and A_(1c)obtained on CE Agilent from reference samples containing differentpurified fractions of glycated haemoglobin using the analysis bufferdescribed in patent U.S. Pat. No. 5,599,433, which contains 100 mM ofCAPS and 300 mM of boric acid (pH: 11; temperature: 24° C.; voltage: 6.1kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

FIG. 3: standard electrophoretic profiles for A_(1a,b) and A_(1c)obtained on CE Agilent from reference samples containing differentpurified fractions of glycated haemoglobin, using a buffer compositioncontaining 200 mM of CAPSO and 10 mM of putrescine but no boratecompound, nor boronate compound (pH: 10.20; temperature: 24° C.;voltage: 6.1 kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

FIG. 4: standard electrophoretic profiles for A_(1a,b) and A_(1c)obtained on CE Agilent from reference samples containing differentpurified fractions of glycated haemoglobin, using a buffer compositioncontaining 200 mM of CAPSO, 10 mM of putrescine and 50 mM de borate (pH:10.20; temperature: 24° C.; voltage: 6.1 kV, i.e. 190 V/cm; injection:50 mbars 20 s).

FIG. 5: standard electrophoretic profiles for A_(1a,b) and A_(1c)obtained on CE Agilent from reference samples containing differentpurified fractions of glycated haemoglobin, using a buffer compositioncontaining 200 mM of CAPSO, 10 mM of putrescine and 50 mM of3-carboxyphenylboronic acid (pH: 10.20; temperature: 24° C.; voltage:6.1 kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

FIG. 6: standard electrophoretic profiles for A_(1a,b) and A_(1c)obtained on CE Agilent from reference samples containing differentpurified fractions of glycated haemoglobin, using a buffer compositioncontaining 200 mM of CAPSO, 10 mM of putrescine and 50 mM of3,5-dicarboxyphenylboronic acid (pH: 10.20; temperature: 24° C.;voltage: 6.1 kV, i.e. 190 V/cm; injection: 50 mbars 20 s).

FIG. 7: standard electrophoretic profiles for A_(1a,b) and A_(1c)obtained on CE Agilent from reference samples containing differentpurified fractions of glycated haemoglobin, using a buffer compositioncontaining 200 mM of CAPSO, 15 mM of putrescine and 100 mM of3,5-dicarboxyphenylboronic acid (pH: 10.20; temperature: 30° C.;voltage: 10 kV, i.e. 310 V/cm; injection: 50 mbars 20 s).

FIG. 8: standard electrophoretic profiles A₀, A_(1a,b) and A_(1c)obtained on CE Agilent from reference samples containing HbA₀ and/ordifferent purified fractions of glycated haemoglobin, using a buffercomposition containing 200 mM of CHES, 20 mM of putrescine and 30 mM of3,5-dicarboxyphenylboronic acid at a pH of 9.40. Fused silica capillary,uncoated (temperature: 34° C.; voltage: 17.3 kV i.e. 520 V/cm; injection50 mbars 20 s).

FIG. 9: standard electrophoretic profiles A₀, A_(1a,b) and A_(1c)obtained on CE Agilent from reference samples containing HbA₀ and/ordifferent purified fractions of glycated haemoglobin, using a buffercomposition containing 200 mM of CHES, 20 mM of putrescine and 30 mM of3,4-dicarboxyphenylboronic acid at a pH of 9.40. Fused silica capillary,uncoated (temperature: 34° C.; voltage: 17.3 kV i.e. 520 V/cm; injection50 mbars 20 s).

FIG. 10: standard electrophoretic profiles for A_(1a,b) and A_(1c)obtained on Capillaries® (Sebia) from reference samples containingdifferent purified fractions of glycated haemoglobin, using a buffercomposition containing 200 mM of CHES buffer, 20 mM of putrescine and 30mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40. Fused silicacapillary, uncoated (temperature: 34° C.; voltage: 9.4 kV, i.e. 520V/cm; injection 20 mbars 6 s).

FIG. 11: electropherogram obtained on Capillaries® (Sebia) from a normalhuman blood sample diluted to ⅙th in the haemolyzing solution (water+1g/L de Triton X100), using a buffer composition containing (A) 200 mM ofCAPSO buffer, 10 mM of putrescine and 50 mM of3,5-dicarboxyphenylboronic acid at a pH of 10.2, or (B) 200 mM of CH ESbuffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronicacid, at a pH of 9.40. Fused silica capillary, uncoated (temperature:34° C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 8 mbars 6 s).

FIG. 12: study of the influence of the concentration of3,5-dicarboxyphenylboronic acid in the buffer composition on theseparation between A_(1c) and A_(o) haemoglobins (temperature: 30° C.;voltage: 10 kV, i.e. 310 V/cm).

FIG. 13: electropherogram obtained on Capillaries® (Sebia) from a normalhuman blood sample comprising a variant HbE, diluted to ⅙^(th) in thehaemolyzing solution (water+1 g/L de Triton X100), using a buffercomposition containing 200 mM of CHES buffer, 20 mM of putrescine and 30mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fusedsilica capillary, uncoated (temperature: 34° C.; voltage: 9.4 kV, i.e.520 V/cm; injection 8 mbars 6 s).

FIG. 14: electropherogram obtained on Capillaries® (Sebia) from acontrol AFSC diluted to ⅙^(th) in the haemolyzing solution (water+1 g/Lde Triton X100), using a buffer composition containing 200 mM of CHESbuffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronicacid, at a pH of 9.40, and a fused silica capillary, uncoated(temperature: 34° C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 8 mbars6 s).

FIG. 15: electropherogram obtained on Capillaries® (Sebia) from a poolof normal blood comprising F and Bart's variants, diluted to ⅙^(th) inthe haemolyzing solution (water+1 g/L de Triton X100), using a buffercomposition containing 200 mM of CHES buffer, 20 mM of putrescine and 30mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fusedsilica capillary, uncoated (temperature: 34° C.; voltage: 9.4 kV, i.e.520 V/cm; injection 8 mbars 6 s).

FIG. 16: electropherogram obtained on Capillaries® (Sebia) from a normalhuman blood sample comprising a HbD variant, diluted to ⅙^(th) in thehaemolyzing solution (water+1 g/L de Triton X100), using a buffercomposition containing 200 mM of CHES buffer, 20 mM of putrescine and 30mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40, and a fusedsilica capillary, uncoated (temperature: 34° C.; voltage: 9.4 kV, i.e.520 V/cm; injection 8 mbars 6 s).

FIG. 17: comparison of the results obtained by the inventors bycapillary electrophoresis using the Capillaries® (Sebia) analyzer withthe results obtained by HPLC with the Variant II Turbo® (Bio-Rad)analyzer.

FIG. 18A: agarose gel of HbA_(1c) carried out on Hydrasys® (Sebia)automated machine. Tracks 1 and 2: weak (5.0%) A_(1c) calibrator andstrong (10.8%) A_(1c) calibrator. Tracks 3 to 9: normal whole bloodincubated for 3 h at 37° C. with glucose at a concentration of 0 g/L(reference; track 3), 1 g/L (track 4), 5 g/L (track 5), 10 g/L (track6), 20 g/L (track 7), 30 g/L (track 8) and 50 g/L (track 9).

FIG. 18B: capillary electrophoresis profiles obtained with theCapillaries® (Sebia) analyzer with the samples of FIG. 18A. The analysiswas not, however, carried out for the sample containing 1 g/L of glucoseas there was no visible difference in the gel (FIG. 18A). The sampleswere diluted to ⅙^(th) in the haemolyzing solution (water+1 g/L deTriton X100), using a buffer composition containing 200 mM of CHESbuffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronicacid, at a pH of 9.40, and a fused silica capillary, uncoated(temperature: 34° C.; voltage: 9.4 kV, i.e. 520 V/cm; injection 8 mbars6 s).

FIG. 18C: table summarizing values for HbA_(1c) obtained on agarose geland with the Capillaries® (Sebia) analyzer for the analyses of the bloodpresented in FIGS. 18A and 18B.

EXAMPLES A. Apparatus and Methods Capillary Electrophoresis

The principle of separation is free solution capillary electrophoresisat an alkaline pH (pH>9), in order to obtain negatively chargedhaemoglobin fractions (the isoelectric point of haemoglobins is in therange 6.05 to 7.63).

The capillary electrophoresis of biological samples was carried out on acapillary electrophoresis apparatus provided with 8 fused silicacapillaries with an internal diameter of 25 microns, a useful length of16 cm and a total length of 18 cm (Capillaries® (Sebia) capillaryelectrophoresis system) or on capillary electrophoresis apparatusprovided with one fused silica capillary with an internal diameter of 25microns, with a useful length of 24 cm and a total length of 32 cm(^(3D)CE capillary electrophoresis system from Agilent Technologies).

Detection was carried out at a wavelength of 425 nm. The blood sampleswere diluted in a haemolyzing solution (Triton X100 1 g/L in water) andinjected by hydrodynamic injection. The capillary was washed before eachanalysis with 0.25 M sodium hydroxide, then with the buffer composition.

Buffer Composition

The buffer compositions in which the capillary electrophoresis wascarried out comprised water, a buffer compound with a pKa in the range 8to 11 (CAPS, CAPSO or CHES depending on the case), a base allowing thepH to be adjusted to the desired value, an optional flow retardant(putrescine), and an optional borate compound (boric acid) or boronate.

3,5-dicarboxyphenylboronic acid (3,5-dCPBA) was obtained from the firmsCombi-blocks Inc. (San Diego, USA) and Apollo Scientific Ltd (Cheshire,United Kingdom).

3,4-dicarboxyphenylboronic acid (3,4-dCPBA) was synthesized by the firmBoroChem SAS (Caen, France).

B. Results Example 1

Capillary electrophoresis was carried out from normal human blood(comprising haemoglobins HbA₀, HbA₁ and HbA₂) diluted to ⅙th in ahaemolyzing solution (1 g/L de Triton X100 dissolved in demineralizedwater), using the analysis buffer described in patent U.S. Pat. No.5,599,433, which contained 100 mM of CAPS and 300 mM of boric acid, pH11. The electropherogram obtained is presented in FIG. 1. The separationbetween the peaks of haemoglobins is poor.

Example 2

Capillary electrophoresis was carried out from reference samples(Exocell, USA), containing different purified fractions of glycatedhaemoglobin (fractions A₀ and A_(1c) or fractions A₀, A_(1b) andA_(1a)), using the analysis buffer described in patent U.S. Pat. No.5,599,433, which contained 100 mM of CAPS and 300 mM of boric acid, pH10.20. The standard electrophoretic profiles for A_(1a,b) and A_(1c)obtained are presented in FIG. 2. The separation between the HbA_(1c)and HbA_(1a,b) haemoglobins is clearly insufficient; the HbA_(1c)electrophoretic peak overlaps the HbA1_(a)/HbA1_(b) peaks.

Example 3

Capillary electrophoresis was carried out from reference samples(Exocell, USA), containing different purified fractions of glycatedhaemoglobin (fractions A₀ and A_(1c) or fractions A₀, A_(1b) andA_(1a)), using a buffer composition containing 200 mM of CAPSO and 10 mMof putrescine (pH10.20) but no borate compound, and also no boronatecompound. The standard electrophoretic profiles for A_(1a,b) and A_(1c)obtained are presented in FIG. 3. The peak corresponding to HbA_(1c)co-migrated with that of HbA_(o).

Example 4

Capillary electrophoresis was carried out from reference samples(Exocell, USA), containing different purified fractions of glycatedhaemoglobin (fractions A₀ and A_(1c) or fractions A₀, A_(1b) andA_(1a)), using a buffer composition containing 200 mM of CAPSO, 10 mM ofputrescine and 50 mM de borate (pH 10.20). The standard electrophoreticprofiles for A_(1a,b) and A_(1c) obtained are presented in FIG. 4. Thepeak corresponding to HbA_(1c) lies between the peaks corresponding toHbA_(o) and HbA_(1a)/HbA_(1b) and is too close to the peak correspondingto other HbA₁s to allow a reliable assay of HbA_(1c).

Example 5

Capillary electrophoresis was carried out from reference samples(Exocell, USA), containing different purified fractions of glycatedhaemoglobin (fractions A₀ and A_(1c) or fractions A₀, A_(1b) andA_(1a)), using a buffer composition containing 200 mM of CAPSO, 10 mM ofputrescine and 50 mM of 3-carboxyphenylboronic acid (pH 10.20). Thestandard electrophoretic profiles for A_(1a,b) and A_(1c) obtained arepresented in FIG. 5. The peak corresponding to HbA_(1c) lies between thepeaks corresponding to HbA_(1b) and HbA_(1a).

Example 6

Capillary electrophoresis was carried out from reference samples(Exocell, USA), containing different purified fractions of glycatedhaemoglobin (fractions A₀ and A_(1c) or fractions A₀, A_(1b) andA_(1a)), using a buffer composition containing 200 mM of CAPSO, 10 mM deDAB and 50 mM of 3,5-dicarboxyphenylboronic acid (pH 10.20). Thestandard electrophoretic profiles for A_(1a,b) and A_(1c) obtained arepresented in FIG. 6. The peak corresponding to HbA_(1c) lies after thepeaks corresponding to HbA_(1b) and HbA_(1a).

Example 7

Capillary electrophoresis was carried out from reference samples(Exocell, USA) containing different purified fractions of glycatedhaemoglobin (fractions A₀ and A_(1c) or fractions A₀, A_(1b) andA_(1a)), using a buffer composition containing 200 mM of CAPSO, 15 mM offlow retardant (putrescine) and 100 mM of 3,5-dicarboxyphenylboronicacid (pH 10.20). The standard electrophoretic profiles for A_(1a,b) andA_(1c) obtained are presented in FIG. 7. The peak corresponding toHbA_(1c) lies after the peaks corresponding to HbA_(1b) and HbA_(1a) andis distinct from these peaks_(;) the separation between the haemoglobinHbA_(1c) and the haemoglobins HbA_(1a) and HbA_(1b) is excellent.

Example 8

Capillary electrophoresis was carried out from reference samplescontaining HbA₀ and/or different purified fractions of glycatedhaemoglobin (fractions A₀ and A_(1c) or fractions A₀, A_(1b) andA_(1a)), using a buffer composition containing 200 mM of CHES, 20 mM offlow retardant (putrescine) and 30 mM of 3,5-dicarboxyphenylboronic acid(at a pH of 9.40). The standard electrophoretic profiles A₀, A_(1a,b)and A_(1c) obtained are presented in FIG. 8. The peak corresponding toHbA_(1c) lies after the peaks corresponding to HbA₀, HbA_(1b) andHbA_(1a) and is clearly distinct from these peaks; the separationbetween the haemoglobin HbA_(1c) and the HbA_(1a) and HbA_(1b)haemoglobins is excellent.

Example 9

Capillary electrophoresis was carried out from reference samplescontaining HbA₀ and/or different purified fractions of glycatedhaemoglobin (fractions A₀ and A_(1c) or fractions A₀, A_(1b) andA_(1a)), using a buffer composition containing 200 mM of CHES, 20 mM offlow retardant (putrescine) and 30 mM of acid 3,4-dicarboxyphenylboronic(at a pH of 9.40). The standard electrophoretic profiles A₀, A_(1a,b)and A_(1c) obtained are presented in FIG. 9. It will be seen that thepeak corresponding to HbA_(1c) lies after the peaks corresponding toHbA₀, HbA_(1b) and HbA_(1a) and is clearly distinct from these peaks_(;)the separation between the haemoglobin HbA_(1c) and the HbA_(1a) andHbA_(1b) haemoglobins is excellent.

By comparing the electrophoretic profiles of examples 8 and 9, it willbe seen that 3,5-dicarboxyphenylboronic acid can produce a slightlybetter result in terms of separation of HbA_(1c) compared with the otherfractions, while 3,4-dicarboxyphenylboronic acid allowed a slightlybetter result to be obtained in terms of focussing.

Example 10

Capillary electrophoresis was carried out on Capillaries® (Sebia) fromreference samples (Exocell, USA) containing different purified fractionsof glycated haemoglobin (fractions A₀ and A_(1c) or fractions A₀, A_(1b)and A_(1a)), using a buffer composition containing 200 mM of CHESbuffer, 20 mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronicacid, at a pH of 9.40. The standard electrophoretic profiles forA1_(a,b) and A_(1c) obtained are presented in FIG. 10. It will be seenthat the peak corresponding to HbA_(1c) lies after the peakscorresponding to HbA_(1b) and HbA_(1a) and is clearly distinct fromthese peaks; the separation between the haemoglobin HbA_(1c) and theHbA_(1a) and HbA_(1b) haemoglobins is excellent.

Example 11

Analyses by capillary electrophoresis were carried out on normal humanblood diluted to ⅙th in haemolyzing solution (water+1 g/L de TritonX100), using a buffer composition containing either 200 mM of CAPSObuffer, 10 mM of putrescine and 50 mM of 3,5-dicarboxyphenylboronicacid, at a pH of 10.20, or 200 mM of CHES buffer, 20 mM of putrescineand 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40. Theelectropherograms obtained on Capillaries® (Sebia) using a fused silicacapillary, uncoated, are presented in FIGS. 11A and 11B respectively. Inboth cases, complete separation of the haemoglobin HbA_(1c) from theother haemoglobins forms is observed.

Example 12

The influence of concentration of 3,5-dicarboxyphenylboronic acid in thebuffer composition on the separation between the A_(1c) and A_(o)haemoglobins was studied. The buffer composition used contained 200 mMof CAPSO, 15 mM of putrescine and 0 to 120 mM of3,5-dicarboxyphenylboronic acid. The results are presented in FIG. 12.The A_(1c)/A_(o) separation increased with the concentration of3,5-dicarboxyphenylboronic acid.

Example 13

Capillary electrophoreses were carried out on Capillaries® (Sebia) usingfour different samples diluted by ⅙th in the haemolyzing solution(water+1 g/L de Triton X100): normal human blood comprising a HbEvariant (FIG. 13), a AFSC control (FIG. 14), a pool of normal bloodcomprising F and Bart's variants (FIG. 15) and normal human bloodcomprising a HbD variant (FIG. 16). Capillary electrophoresis wascarried out in a buffer composition containing 200 mM of CHES buffer, 20mM of putrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pHof 9.40, using a fused silica capillary, uncoated.

FIGS. 13-16 show the absence of interference from the principal variantsof haemoglobin (E, F, S, C, D and Bart) with the HbA_(1c) fraction.Note, however, that in the case of Bart's haemoglobin, the resolution isnot complete between the Hb Bart and HbA_(1c) fractions. As aconsequence, in order to be able to assay HbA_(1c) in the presence of HbBart, these two fractions should be capable of being quantified using asuitable integration method. If this is not possible, it will benecessary to alert the user to this, in case he observed this type ofprofile with a shoulder on the expected peak.

Example 14

The results obtained by capillary electrophoresis by the method ofinvention using the Capillaries® (Sebia) analyzer were compared with theresults obtained with one of the reference techniques: HPLC with theVariant II Turbo® (Bio-Rad).analyzer.

Capillary electrophoresis was carried out from whole blood diluted to⅙th in the haemolyzing solution (water+1 g/L de Triton X100), in abuffer composition containing 200 mM of CHES buffer, 20 mM of putrescineand 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40.

FIG. 17 shows the very good correlation of this novel technique foranalysis by capillary electrophoresis with the analysis of HbA_(1c) byHPLC with the Variant II turbo® from Bio-Rad. After calibration of theEC data using 2 calibrators (weak A1_(c) and strong A1_(c)), the valuesobtained by the method of invention were very close to those obtained bythe reference method.

Example 15

Study demonstrating the absence of interference of the labile fractionof HbA_(1c) with the assay of HbA_(1c) using the method of theinvention.

Hypothesizing that the compound complexing glucose and providingnegative charges at an alkaline pH used in the context of the analysismethod of the invention would be capable of interacting with bloodglucose (this interaction is hypothetical and has not beendemonstrated), a study of any interference of free glucose on the resultof the blood of HbA_(1c) was carried out as follows: normal blood wasincubated for 3 hours at 37° C. with different concentrations of glucose(0 to 50 g/L) in order to create the labile form of HbA_(1c) (formobtained before rearrangement of the molecule (Amadori rearrangement)).Once incubation had been carried out, the blood samples were centrifugedand the pellets obtained were reconstituted in physiological water andthe haemolyzing solution (15 μL of pellet+25 μL physiological water+160μL of haemolyzing solution (water+1 g/L Triton X100)) was then analyzedin parallel using a Hydrasys® automated machine from Sebia (HbA_(1c)gel) and using the Capillaries® (Sebia) technique, using a buffercomposition containing 200 mM of CHES buffer, 20 mM of putrescine and 30mM of 3,5-dicarboxyphenylboronic acid, at a pH of 9.40.

The HbA1c gel obtained by the analysis on the Hydrasys® automatedmachine (see FIG. 18A) confirmed the formation of labile fraction of theHbA1c migrating to the same level as the HbA1c and in an increasingconcentration as the concentration of glucose increased duringincubation with the blood. It should be noted that in gel, under thenormal conditions of use defined by Sebia, the labile fraction did notappear, in particular because of the acid pH of the haemolyzingsolution.

In contrast, the analyses carried out on Capillaries® (Sebia) with abuffer composition of the invention (200 mM of CHES buffer, 20 mM ofputrescine and 30 mM of 3,5-dicarboxyphenylboronic acid, at a pH of9.40), on the same blood samples showed that the assay was not perturbedby the presence of free glucose, regardless of the concentration ofincubated glucose, in the range studied (0 to 50 g/L): the profiles andthe values of HbA1c were unchanged (see FIGS. 18B and 18C).

REFERENCES

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1. A method for analysis by capillary electrophoresis of one or severalglycated haemoglobin(s) comprising at least one globin chain, inparticular a beta chain, comprising a glucose residue bound to the aminoacid in the N-terminal position, contained in a biological samplecomprising haemoglobins, said method comprising using a buffercomposition comprising at least one compound which is capable ofspecifically complexing glucose residues of glycated haemoglobins of thebiological sample, in particular the glucose residue(s) bound to anamino acid in the N-terminal position in glycated haemoglobins, and ofproviding said glycated haemoglobin(s) with several negative electriccharges at an alkaline pH.
 2. A method according to claim 1, for theanalysis by capillary electrophoresis of haemoglobin A_(1c) contained ina biological sample comprising haemoglobins.
 3. A method according toclaim 1 or 2, in which said compound which is capable of specificallycomplexing glucose residues of glycated haemoglobins of the biologicalsample and of providing negative charges at an alkaline pH comprises twoor more than two functional groups, at least one of said functionalgroups specifically complexing one or several glucose residue(s), theother, one of the others or all of the other functional group(s)providing said glycated haemoglobin(s) with one or several negativeelectric charge(s) at an alkaline pH.
 4. A method according to any oneof claims 1 to 3, in which the compound which is capable of specificallycomplexing glucose residues of glycated haemoglobins of the biologicalsample and of providing negative charges at an alkaline pH comprises oneor several groups which are anionisables at an alkaline pH, inparticular one or several carboxylate(s), carboxyl(s), sulphonate(s)and/or sulphonyl(s).
 5. A method according to any one of claims 1 to 4,in which the compound which is capable of specifically complexingglucose residues of glycated haemoglobins of the biological sample andof providing negative charges at an alkaline pH comprises one (or more)boronyl and/or boronate group(s), and in particular is: (i) a boronatecompound with general formula RB(OH)₂ or RB(OH)₃ ⁻, in which the group Rcomprises at least one aryl and/or alkyl (linear, branched or cyclic)and/or an aralkyl and/or other functional groups or heteroatoms, and/ora combination thereof, and said group R provides glycated haemoglobinswith one or several negative electric charge(s) at an alkaline pH foreach glucose residue complexed with the boronate group; or (ii) a saltof said boronate compound.
 6. A method according to claim 5, in whichthe boronate compound is a polysubstituted phenylboronate, in particulara phenylboronate which is disubstituted with carboxyl and/or sulphonylgroups, for example, and more particularly a compound in which the groupR is a diacid, preferably a dicarboxylic acid.
 7. A method according toclaim 5 or 6, in which the boronate compound is a dicarboxyphenylboronicacid, preferably selected from 3,4-dicarboxyphenylboronic acid and3,5-dicarboxyphenylboronic acid more preferably3,5-dicarboxyphenylboronic acid.
 8. A method according to any one ofclaims 1 to 7, in which the concentration in the buffer composition ofthe compound which is capable of specifically complexing glucoseresidues of glycated haemoglobin(s) of the biological sample and ofproviding negative charges at an alkaline pH is in stoechiometric excesswith respect to the total quantity of proteins, compared with the totalquantity of all of the haemoglobins present in the biological sample orcompared with the total quantity of all of the haemoglobins comprisingglucose present in the biological sample.
 9. A method according to anyone of claims 1 to 8, in which the concentration in the buffercomposition of the compound which is capable of specifically complexingglucose residues of glycated haemoglobins of the biological sample andof providing negative charges at an alkaline pH is in the range 0.10 to100 mM, preferably in the range 10 to 60 mM, and more preferably in therange 20 to 50 mM, for example 30 mM.
 10. A method according to any oneof claims 1 to 9, in which the buffer composition further comprises aflow retardant, in particular a flow retardant which is an aliphaticdiamine, more particularly an aliphatic diamine selected from1,3-diaminopropane, 1,4-diaminobutane (putrescine), 1,5-diaminopentane(cadaverine), 1-6-diaminohexane, spermine and DETA, or a derivative ofsaid aliphatic diamine, for example an aliphatic polyamine, a cyclicpolyamine or an aliphatic diamine salt, or one of their mixtures, andstill more particularly a flow retardant which is putrescine or aderivative.
 11. A method according to claim 10, in which theconcentration of flow retardant in the buffer composition is in therange 0.10 to 40 mM, preferably in the range 10 to 30 mM and morepreferably in the range 15 to 25 mM, for example 20 mM.
 12. A methodaccording to any one of claims 1 to 11, in which the buffer compositionfurther comprises: a buffer compound having a pKa in the range 8.0 to11.0; and/or a base; and/or a salt, in particular a sodium salt, forexample sodium chloride; and/or an appropriate diluting solution, forexample water.
 13. A method according to claim 12, in which the buffercompound is a zwitterionic compound, in particular a compound selectedfrom AMP, AMPD, AMPSO, bicine, CABS, CAPS, CAPSO, CHES, HEPBS,methylamine, TABS, TAPS, taurine, tricine and Tris, for example CHES,CAPS or CAPSO.
 14. A method according to claim 12 or 13, in which theconcentration of buffer compound in the buffer composition is in therange 20 to 500 mM, preferably in the range 50 to 400 mM, and morepreferably in the range 150 to 300 mM, for example 200 mM.
 15. A methodaccording to any one of claims 1 to 14, in which the buffer compositionhas a pH of 9 or more, preferably in the range 9.0 to 11.0 and morepreferably a pH in the range 9.0 to 10.0, for example a pH of 9.4.
 16. Amethod according to any one of claims 1 to 15, comprising the followingsteps: a) introducing the buffer composition and biological sample intoan electrophoresis capillary; and b) separating the constituents of thebiological sample by capillary electrophoresis.
 17. A method accordingto claim 16, in which the step for separating the constituents of thebiological sample is followed by a step for detection of one or severalhaemoglobin(s) present(s) in the biological sample.
 18. A methodaccording to claim 16 or 17, further comprising a step for generating anelectropherogram from a detection signal which is proportional to thequantity of haemoglobin(s) detected.
 19. A method according to any oneof claims 1 to 18, further comprising a step for determining, inparticular from an electropherogram as defined in claim 18, the quantityof one or several haemoglobin(s) present in the biological sample and/orof the proportion of one or several haemoglobin(s) present in thebiological sample with respect to the total quantity of proteins, thetotal quantity of haemoglobin or the quantity of certain haemoglobinspresent in the biological sample.
 20. A method according to any one ofclaims 1 to 19, further comprising a step for quantification of one orseveral haemoglobin(s) present in the biological sample compared withone or several standardized calibrator(s).
 21. A method according to anyone of claims 1 to 20, in which the biological sample is a blood sample,in particular a normal or non-normal blood sample, washed, decanted,centrifuged or whole, said sample being haemolysed if appropriate.
 22. Amethod according to any one of claims 1 to 21, in which the biologicalsample is diluted in a haemolyzing solution, in particular a haemolyzingsolution selected from the group constituted by: the haemolyzingsolution Capillaries® Hemoglobin(e); the haemolyzing solution Hydragel®HbA_(1c); the haemolyzing solution MINICAP® Hemoglobin(e); pure water;water supplemented with surfactant, in particular water supplementedwith Triton; and mixtures thereof.
 23. A buffer composition appropriatefor the analysis, by capillary electrophoresis, of one or severalglycated haemoglobins comprising a glucose residue bound to the aminoacid in the N-terminal position on at least one globin chain and inparticular on the beta globin chains, contained in a biological samplecomprising one or several haemoglobin(s), said composition being asdefined in any one of claims 1 to
 15. 24. A kit comprising a buffercomposition according to claim 23 and instructions for use ifappropriate.
 25. A compound which is capable of specifically complexingglucose residues of glycated haemoglobins of a biological sample and ofproviding negative charges at an alkaline pH, as defined in any one ofclaims 1 to 7 and/or a buffer composition as defined in any one of claim1 to 15 or 23 and/or a kit according to claim 24, for use in thediagnosis of diabetes in a human or non-human mammal and/or to monitorthe glycaemic balance in a human or non-human mammal.
 26. A compoundwhich is capable of specifically complexing glucose residues of glycatedhaemoglobins of a biological sample and of providing negative charges atan alkaline pH, for use according to claim 25, wherein said compound is:(i) a boronate compound with general formula RB(OH)₂ or RB(OH)₃ ⁻, inwhich the group R comprises at least one aryl and/or alkyl (linear,branched or cyclic) and/or an aralkyl and/or other functional groups orheteroatoms, and/or a combination thereof, and said group R providesglycated haemoglobins with one or several negative electric charge(s) atan alkaline pH for each glucose residue complexed with the boronategroup; or (ii) a salt of said boronate compound.
 27. Use of a compoundwhich is capable of specifically complexing glucose residues of glycatedhaemoglobins of a biological sample and of providing negative charges atan alkaline pH, as defined in any one of claims 1 to 7 and/or of abuffer composition as defined in any one of claim 1 to 15 or 23, and/orof a kit according to claim 24, for the separation, by capillaryelectrophoresis, of one or several glycated haemoglobin(s) comprising atleast one globin chain comprising a glucose residue bound to the aminoacid in the N-terminal position, from other proteins, in particular fromat least one other haemoglobin and preferably from the otherhaemoglobins present in a biological sample.